Methods and kits useful for the simplification of complex peptide mixtures

ABSTRACT

Methods and reagents for obtaining simplified mixtures of peptides from a sample containing a number of peptides are disclosed. The simplified sample can be easier to analyze than the original peptide sample yet it is representative of all or nearly all of the proteins present in the mixed protein sample from which the original and more complex peptide sample was derived. The methods entail the use of tagging moieties that include an amino-acid-specific reactive group (R). The tagging moieties “tag” peptides or proteins at specific amino acids (e.g., by reacting with an amino acid to form a covalent bond), ultimately allowing the isolation of peptides that contain those specific amino acids. Other methods entail the used of a reactive moiety (R P ) that comprises a reagent that selectively interacts with selected proteins, either covalently or noncovalently. For example, R P  can be a natural ligand for a receptor that is to be tagged or a protein that interacts with a second protein that is to be tagged. It can be an enzymatic substrate or other element of molecular recognition such as an antibody, ATP, GTP, NAD, NADP, NADH, NADPH, ubiquitin, or structural analogs thereof.

RELATED APPLICATION INFORMATION

[0001] This application claims priority from provisional applicationserial No. 60/281,244, filed Apr. 3, 2001, hereby incorporated byreference.

BACKGROUND OF THE INVENTION

[0002] Proteomic techniques that permit the identification,quantification, and localization of proteins in cells will advance theunderstanding of cell function and development far beyond what has beenachieved by genomic techniques. For example, the ability of advancedmass spectrometry techniques to analyze complex protein mixtures, e.g.,multi-protein complexes, cell fractions and whole cells extracts,promises to provide powerful high throughput diagnostic and screeningmethods.

[0003] Mass spectrometry can be used to identify single proteins orlarge number of proteins in mixtures. In addition, mass spectrometry canbe used to sequence a peptide de novo. For example, tandem massspectrometry of peptides generated by proteolytic digestion of a complexprotein mixture (e.g., a cell extract) can be used to identify andquantify the proteins present in original mixture. This result can beachieved because tandem mass spectrometers capable of selecting singlem/z values and subjecting the ions to collision induced disassociation(CID) can be used to sequence and identify peptides. The informationcreated by CID of a peptide can be used to search protein and nucleotidesequence databases to identify the amino acid sequence represented bythe spectrum and thus identify the protein from which the peptide wasderived.

[0004] Tandem mass spectrometry used to identify a peptide in a complexmixture of peptides derived from digested proteins utilizes three typesof information. First, the mass of the peptide is obtained. Thisinformation alone can greatly reduce number of possible peptidesequences, particularly if the protein was digested with a sequencespecific protease. The second type of information is the pattern offragment ions produced by CID of the peptide ion. Analytical methodsthat compare the fragment ion pattern to theoretical fragment ionpatterns generated computationally from sequence databases can be usedto identify the peptide sequence. Such methods can identify the bestmatch peptides and statistically determine which peptide sequence ismore likely to be correct. The accuracy of the predictions can beincreased further by using multiple dimensions of MS analysis to obtainde novo the sequence of a portion of a peptide. This direct sequenceinformation can be used to further increase the accuracy of theprediction based on the fragment ion patterns. Once the peptide isidentified, the protein from which it was generated can be readilydetermined by searching sequence databases.

[0005] Proteins in complex mixtures, e.g., cell extracts, can beidentified by a combination of enzymatic proteolysis, liquidchromatographic separation, tandem mass spectrometry, computeralgorithms which correlate peptide mass spectra to those theoreticallypredicted based on sequence databases and by de novo sequencing.

[0006] Electrospray ionization permits liquid chromatography to bedirectly coupled to a tandem mass spectrometer so that complex mixturescan be temporally separated prior to introduction into the massspectrometer. The increase in the number of organisms for which acomplete genome sequence is available will greatly increase the value ofthis approach to the analysis of complex mixtures.

SUMMARY OF THE INVENTION

[0007] The invention features methods and reagents for obtainingsimplified mixtures of peptides from a sample containing a number ofpeptides, e.g., a sample created by proteolytic digestion of a mixtureof proteins, e.g., a mixture of proteins obtained from a biologicalsample. The methods and reagents of the invention can be used todecrease the number of peptides (or proteins) in the sample according toa rational and controlled scheme so as to obtain a simplified peptidesample containing fewer peptides. For example, starting with a peptidesample created by proteolytic digestion of a mixed protein sample, onecan obtain a simplified peptide sample that contains only one or a fewof the peptides created by proteolytic digestion of each the proteins inthe mixed protein sample. The simplified sample can be easier to analyzethan the original peptide sample yet it is representative of all ornearly all of the proteins present in the mixed protein sample fromwhich the original and more complex peptide sample was derived.Accordingly, the simplified peptide mixture can be used to identify andquantify all or nearly all of the proteins in the original mixed proteinsample. The simplified mixture is useful even when it does not includeat least one peptide from each of the proteins in the mixed proteinssample since in some cases it is not necessary to identify or quantifyall of the proteins in the mixed protein sample.

[0008] The methods and compositions of the invention are useful foranalyzing peptides that are generated by the enzymatic digestion ofcomplex protein mixtures (e.g., cell extracts). The methods andcompositions of the invention are useful in any setting in which it isdesirable to reduce the complexity of a peptide mixture in a controlledand specific manner and find particular application in the preparationof peptide samples for analysis by mass spectrometry.

[0009] The methods of the invention entail the use of tagging moietiesthat include an amino-acid-specific reactive group (R). The taggingmoieties “tag” peptides or proteins at specific amino acids (e.g., byreacting with an amino acid to form a covalent bond), ultimatelyallowing the isolation of peptides that contain those specific aminoacids. The amino acid tagged by a given tagging moieties depends on theidentity of the R group present on the moiety. One R group (R_(S/T))tags serines (ser) and threonines (thr) when they are present at theamino terminus of a peptide. Another R group (R_(C)) tags cysteines(cys), present anywhere in a peptide, at its thiol group. Another Rgroup (R_(L)) tags lysines (lys), present anywhere in a peptide, at itsprimary amine

[0010] The invention also features a reactive moiety (R_(P)) thatcomprises a reagent that selectively interacts with selected proteins,either covalently or noncovalently. For example, R_(P) can be a naturalligand for a receptor that is to be tagged or a protein that interactswith a second protein that is to be tagged. It can be an enzymaticsubstrate or other element of molecular recognition such as an antibody,ATP, GTP, NAD, NADP, NADH, NADPH, ubiquitin, or structural analogsthereof. R_(P) is a special case of R as its use is intended to simplifypeptide samples by selectively reducing the number of proteins appearingin the mixed protein sample prior to proteolysis.

[0011] The reactive group of the tagging moiety is directly orindirectly associated with other groups that facilitate the isolation oftagged peptides. Thus, the tagging moiety can include a linker group (L)which can connect the R group to a group (B or M) that facilitates thecapture of tagged peptides. The B group is a group, e.g., biotin thatcan selectively bind to a capture reagent, e.g., strepavidin. The Mgroup is a magnetic particle that can be attracted by a magnetic force.

[0012] The isolated peptides can be analyzed by mass spectrometry or anyother desired method. For example, Mass spectrometry can be used toidentify and/or quantify one or more of the peptides in the simplifiedmixture.

[0013] Thus, the invention features a method for reducing the number ofpeptides present in a sample, the method comprising: (a) providing atagging moiety comprising three covalently-connected parts:

R-L-B

[0014] wherein R is a reactive group (for example: R_(S/T), R_(C), R_(L)or R_(P)) that reacts with peptides or proteins; L is a linker groupcontaining zero or more atoms in a straight or branched chain and anoptional selective cleavage site; and B is a group that can selectivelybind to a capture reagent; (b) reacting the sample with the taggingmoiety to provide tagged peptides or proteins; (c) contacting the taggedpeptides or proteins with a capture reagent to provide captured taggedpeptides or proteins and isolating the captured tagged peptides orproteins from other material in the sample (e.g., non-tagged peptides orproteins); (d) releasing at least the peptide portion or protein portionof the tagged peptide or protein from the capture reagent to providereleased modified peptides or proteins; and (e) analyzing the releasedmodified peptides or proteins, or fragments thereof, by massspectrometry to identify at least one peptide or protein present in thesample. The released modified peptide or protein can include all or partof R and all or part or none of L.

[0015] In addition, the invention optionally features the moiety R-L*-Bwhere L* is isotopically labeled version of L. Such isotopically labeledtagging moieties provide a means for quantifying two or more sampleseach labeled differently from each other and subsequently mixed togetherprior to mass analysis. The isotopic label can be ²H, ¹³C, ¹⁵N, ¹⁸O,³⁴S, or any other suitable isotopic label. The invention also optionallyfeatures R*-L-B and R-L-B* were B* is an isotopically labeled version ofB and R* is an isotopically labeled version of R. The moiety can alsoprovide a means for obtaining differentially isotopically labeledpeptides or proteins.

[0016] Elsewhere in this patent application, “peptide” can be read toinclude “protein” or “modified peptides or proteins”.

[0017] In other embodiments, proteins or peptides can be captured foranalysis by mass spectrometry by using a tagging moiety comprising threecovalently-connected parts:

M-L-R

[0018] wherein M is a magnetic particle; L is a linker that iscovalently attached to M and contains zero or more atoms in a straightor branched chain and an optional selective cleavage site; and R is aspecific reactive group including one of the reactive groups specifiedabove (R_(S), R_(C), R_(L), or R_(P)) that reacts with peptides orproteins. In this embodiment, tagged peptides or proteins may beisolated from untagged peptides or proteins by selective application ofmagnetic force on the M moiety, rather than by selective reagent captureof a B moiety.

[0019] Peptides can derive from enzymatically-digested proteins or beprocessed in other biological or synthetic means. Proteins can beisolated from, e.g., a patient cell sample, a patient serum sample, or apatient tissue sample. The proteins can be derived from, e.g., culturedcells, cultured cells treated with a compound of interest (e.g., atherapeutic compound or a potential therapeutic compound), or plantcells, microbial cells, a virus, or genetically modified cells.

[0020] In various embodiments R_(c) comprises a thiol specific reactivegroup (e.g. a maleimide group or a pyridyl-dithio group), R_(L)comprises an amine specific reactive group (e.g. a succinimide group),and R_(S/T) comprises a Thr/Ser specific reactive group (e.g. ahydrazide group). R_(P) may be comprised of R_(S/T), R_(C), R_(L) or anenzymatic substrate or other element of molecular recognition such asantibodies, or ATP, GTP, NAD, NADP, NADH, NADPH, ubiquitin, orstructural analogs thereof.

[0021] L is a single or multipart linker that may be composed ofbiological or nonbiological oligomeric structure. For example, L cancomprise a polypeptide chain of any sequence, a chain of identical aminoacids (e.g., poly-glycine or poly-alanine), a chain of alternating aminoacids or a chain of various amino acids. L can include, for example: O,S, NH, CO, COO, COS, S—S, CH₂, an alkyl group, an alkenyl group, andalkynyl group an alkoxy group, or an aryl group. L may contain chemicalor enzymatic cleavage sites to enable the release of modified peptide orprotein from M. L may or may not be differentially labeled with stableor radioactive isotope atoms.

[0022] In certain embodiments L is cleavable and contains a disulfidegroup or a vicinal diol group, or an ortho-nitrobenzyl ether, and incertain embodiments L is isotopically labeled, and/or R is isotopicallylabeled.

[0023] A variety of cleavage sites, either chemical or enzymatic orboth, can be included in L. For example chemical cleavage there can be adisulfide bond that is cleaved using a suitable reducing agent. A glycolor diol bond can be cleaved by oxidation. A diazo bond can be cleavedusing dithionite. An ester can be cleaved using hydroxylamine, acid, orbase. A sulfone can be cleaved using a suitable base. Where L includes apolypeptide, it can be cleaved using a protease. A glycerol ester can becleaved using a lipase. A phospho-ester can be cleaved usingphosphatase. Polynucleotides or oligonucleotides can be cleaved using anuclease.

[0024] In various embodiments the releasing step comprises exposing thecaptured tagged polypeptides to reducing agents or other cleavagereagents, and the released modified peptides are separated bychromatography prior to analysis by mass spectrometry. In otherembodiments the captured tagged peptides are first treated to release Bfrom the capture reagent (or to release M from capture from a magneticfield) and then treated to release the modified peptide or proteinportion from B and all or part of L (or from M and all or part of L).

[0025] The M, L, and R moieties can be connected synthetically in anumber of ways. For example, commercially available magnetic particleswith the structure M-L-NH₂ can be purchased. Analogously, commerciallyavailable capture groups with the structure B- L-NH₂ may be purchased.The amino group can be reacted with various bifunctional cross-linkingagents so as to create various R groups attached to either M or Bthrough L.

[0026] Tagging moieties in which R is covalently attached to a magneticparticle have several advantages. First, they can be used to capturepeptides in a single step. This allows for greater efficiency and easeof sample handling compared to methods in which peptides are firsttagged with a reagent that includes an affinity label followed bycapture of the affinity label on a solid support, e.g., a bead or solidparticle, that is coated with a capture reagent that binds to theaffinity label. Tagging moieties in which R is covalently attached to amagnetic particle or the solid phase material avoids the need forcarrying out two binding steps in order to link the captured peptide tothe solid support. In addition, by using a solid support, e.g., a M-L-Rstructure, sample clean up and removal of non-derivitized peptides canbe accomplished in a single step. The capture can thus be faster andmore efficient. Moreover, since the tagged peptides are isolated using amagnetic force to attract the magnetic particle, the capture step is notone that is subject to interference by components present in thereaction mixture, e.g., peptides, impurities, and is unaffected by suchfactors as buffer conditions and temperate. Moreover, magnetic particlesprovide many advantages in ease of sample handling. A suitable taggingmoiety of the form M-L-R can be prepared as follows. A magnetic particle(the M portion) having a covalently-bound primary amino group Dynal A/S(Oslo, Norway) can be activated by reaction withN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), aheterobifunctional crosslinker available from Pierce Chemical Company.This reagent provides a pyridine thiol group that can be displaced bythe thiol of a thiol-containing peptide. The peptide may also be asynthetic peptide that also includes a primary amino group to which adifferent R group may be attached. Thus, the thiol-containing peptidemakes up a portion of the L group. The thiol-containing peptide can belabeled, e.g., with ¹³C or deuterium. In such cases, the M-L-R moiety isdesignated as M-L*-R. The isotope labeling allows for the relativequantification of peptides or proteins in different samples that aremixed together and analyzed by mass spectrometry simultaneously. Thethiol-containing peptide preferably contains a cleavage site to allowfor release of the captured peptide. Examples of cleavage sites includethose with disulfide groups, that allow for chemical cleavage, or groupsthat allow for enzymatic cleavage, e.g., by trypsin. Peptides aredesirable L groups, in part, because they are rather easy and relativelysimple to synthesize. Because they can be designed to be substantiallyhydrophilic, substantially hydrophobic, or neither, they can be adaptedto a variety of solution conditions. Moreover, peptides have structuraland conformational flexibility. A peptide can be readily designed toinclude chemical cleavage site, an enzymatic cleavage site or both typesof cleavage sites.

[0027] As noted above, the primary amino group of the thiol-containingpeptide can react with various moieties to create varying R portions tothe structure M-L-R (or B-L-R). For example, the primary amino group canreact with a moiety including a hydrazide group. In this case, the Rmoiety will be selective for peptides with the threonine or serine atthe amino terminus. Alternatively, the primary amino group can reactwith a moiety containing a malemide group. In this case, the R moietywill react selectively with cysteine-containing peptides. Otherelectrophilic R groups suitable for reacting with cysteine-containingpeptides include: epoxides, α-haloacyl, nitriles, sulfonated alkylthiols, and sulfonated aryl thiols. The R moiety can also include asuccinimide group for reaction with an amino group (e.g., lysine).

[0028] The M-L-R or B-L-R moiety can be used to react with: (1)peptides, including those arising from enzymatic digestion of proteins;(2) proteins in a native form; (3) proteins in a denatured form and (4)proteins in their native, membrane-embedded form.

[0029] In the case of proteins in a native form (and certain peptideslarge enough to assume a secondary structure), only those specific aminoacids that are presented (i.e., sterically available) on the outer partof the molecule will react with the M-L-R or B-L-R moiety. Therefore,the attachment allows for the specific targeting of “presented” parts ofthe protein. In the case of denatured protein, all parts of the proteinare potentially accessible. The protein may subsequently be digestedinto peptide components. An M-L-R moiety can be used to capture aprotein in its native or denatured form. The protein can then bedigested chemically or enzymatically. The M-L-R moieties (with attachedpeptides) can be washed to remove unbound peptides (and other unwantedmaterial). Subsequently, the modified peptides are released. In somecases it may be desirable to conduct more than one washing step. Forexample, tagged peptides or tagged polypeptides can be washed before orafter capture or both.

[0030] In the case of tagging polypeptides or proteins prior toenzymatic or chemical digestion several approaches are possible. Forexample, a portion of a polypeptide present in a sample can be capturedan analyzed by: (a) providing a tagging moiety having the formula:R-L-M, wherein R is a reactive group that reacts with polypeptidescomprising a selected amino acid, L is a linker group, and M is amagnetic particle that can be attracted by a magnetic force; (b)reacting the sample with the tagging moiety to provide a taggedpolypeptide; (c) isolating the tagged polypeptide by applying a magneticforce that attracts M to provide an isolated tagged polypeptide; (d)enzymatically or chemically digesting the isolated tagged polypeptide toprovide an isolated tagged polypeptide fragment; (e) releasing at leastthe polypeptide fragment portion of the isolated tagged polypeptidefragment from the M group to provide a released modified polypeptidefragment; and (f) analyzing the released modified polypeptide fragmentby mass spectrometry. In this method the digestion step takes placeafter the tagged polypeptides have been isolated. The order of the stepscan be changed so that the digestion takes place prior to isolation ofthe tagged polypeptides. In this approach the method includes: (a)providing a tagging moiety having the formula: R-L-M, wherein R is areactive group that reacts with polypeptides comprising a selected aminoacid, L is a linker group, and M is a magnetic particle that can beattracted by a magnetic force; (b) reacting the sample with the taggingmoiety to provide a tagged polypeptide; (c) digesting the taggedpolypeptide provide an tagged polypeptide fragment; (d) isolating thetagged polypeptide fragment by applying a magnetic force that attracts Mto provide an isolated tagged polypeptide fragment; (e) releasing atleast the polypeptide fragment portion of the isolated taggedpolypeptide fragment from the M group to provide a released modifiedpolypeptide fragment; and (f) analyzing the released modifiedpolypeptide fragment by mass spectrometry.

[0031] Intact proteins or polypeptides can also be tagged using an R-L-Btagging moiety. Thus, the invention includes a method comprising (a)providing a tagging moiety having the formula: R-L-B, wherein R is areactive group that reacts with polypeptides comprising a selected aminoacid, L is a linker group, and B is a group that can selectively bind toa capture reagent; (b) reacting the sample with the tagging moiety toprovide a tagged polypeptide; (c) contacting the tagged polypeptidefragment with the capture reagent to provide a captured taggedpolypeptide; (d) digesting the captured tagged polypeptide to provide acaptured tagged polypeptide fragment; (e) releasing at least thepolypeptide portion of the capture tagged polypeptide fragment from theB group to provide released modified polypeptide fragment; and (f)analyzing the released modified polypeptide fragment by massspectrometry. The invention also includes a method comprising: (a)providing a tagging moiety having the formula: R-L-B, wherein R is areactive group that reacts with polypeptides comprising a selected aminoacid, L is a linker group, and B is a group that can selectively bindsto a capture reagent; (b) reacting the sample with the tagging moiety toprovide a tagged polypeptide; (c) digesting the tagged polypeptideprovide an tagged polypeptide fragment; (d) contacting the taggedpolypeptide fragment with the capture reagent to provide a capturedtagged polypeptide fragment; (e) releasing at least the polypeptidefragment portion of the captured tagged polypeptide fragment from the Bgroup to provide a released modified polypeptide fragment; and (f)analyzing the released modified polypeptide fragment by massspectrometry.

[0032] When polypeptides or proteins are tagged using one of the taggingmoieties of the invention, it should be understood that the polypeptideor protein is not necessarily an intact, naturally occurring polypeptideor protein (although it may be). In some a naturally occurring orpolypeptide can be subjected to preliminary treatment that reduces itsize before it is tagged.

[0033] In another embodiment specifically to address membrane embeddedand/or insoluble proteins, a tagging moiety comprising the structuralgroup M, L, Sol, and R can be used for simplifying protein and peptidemixtures for analysis by mass spectrometry. In this tagging moiety M isa magnetic particle, L is a linker group, Sol is a membrane-impermeablesolublizer group and R is a chemically reactive group that canselectively bind with specific amino acids or modified amino acids, suchas those described above. The solublizer group can be a polymericspecies, such as polyethylene glycol (PEG) or methoxylated polyethyleneglycol (MPEG), that enhances solubility of protein that is linked to theR group. This approach is particularly useful when the protein itself isnot be sufficiently soluble in aqueous solution once removed from itsmembrane. Various arrangements of these components can be usedincluding:

M-L-Sol-R,

M-Sol-L-R,

M-L-Sol-L-R,

M-L-R-Sol,

M-L-R-L-Sol, and

M-L-R

□

Sol

[0034] One or more chemical cleavage sites (e.g., as described above)may be provided between the M, Sol or R groups, but in the mostpreferred embodiments the L group includes a cleavage site near themagnetic particle to yield, after cleavage, the peptide for proteinlinked to R, L and Sol. The cleavage site may be comprised of adisulfide or an enzyme-cleavable oligo-peptide. Thus, after cleavage theSol group enhances the aqueous solubility of bound protein. The enhancedsolubility may be beneficial for the processes to which the protein mustbe exposed prior to analysis by mass spectrometry utilizing fluidionization techniques such as electrospray. A second cleavage site canbe used to release the Sol group leaving the peptide or protein linkedto all or part of R and all or part or none of L.

[0035] Optionally, one or more of L, R and Sol may be isotopicallylabeled.

[0036] As discussed above, a B group can be used in place of an M group.Thus, biotin or some other affinity base can be used in place of amagnetic particle. Under these circumstances, the tagged peptide can becaptured using, for example, a streptavidin-coated magnetic particle. Inthis case, the solubilizer consisting of R, L, Sol and B also can playthe function of preventing transfer of the amino acid-specific reagent(R) across lipid membranes. Thus, the agent is effective to selectspecific intramembrane proteins that present an aspect to the exteriorof the lipid membrane and, in addition, solubilizes these proteinsduring the proteolytic and other sample preparation steps upstream ofLCMS analysis.

[0037] There are also applications for a general solubilizing agentconsisting of R, L, and Sol for generally solubilizing membrane-boundprotins. A solid-phase capture is not always required. For example,various liquid chromatographic means can be used to isolate themembrane-derived components.

[0038] The invention also features reagent kits comprising taggingmoieties having the formulae:

R-L-B

R-L-M

R, L, Sol and M

R, L, Sol, and B, and

R, L, and Sol

[0039] wherein R includes one of the above four reactive groups(R_(S/T), R_(C), R_(L), or R_(P)) as described in detail above, L is alinker group as described above, B is a group that can selectively binda capture reagent as described above; and optionally a proteolyticenzyme. In various embodiments L or R is isotopically labeled (denotedL* or R*), R_(S) contains a structure of the formula —CO—NH—NH₂, and theentire tagging moiety is biotin hydrazide.

[0040] In other embodiments the reagent kit further comprises: a capturereagent, a capture reagent comprising avidin or streptavidin bound to asolid support such as a latex particle or magnetic particle; D-biotin;an oxidizing agent (e.g., sodium metaperiodate) and an agent capable ofquenching the oxidizing agent, and buffers formulated specifically tooptimize the reactions and separations involved

[0041] In other embodiments, the analysis by mass spectrometry comprisesdetermining the molecular weight of at least one released modifiedpeptide and/or the amino acid sequence of at least a portion of at leastone released modified peptide and the peptides are treated chemicallyprior to reacting with the tagging moiety.

[0042] The methods of the invention are useful, in part, because theanalysis of complex peptide mixtures can be very difficult and timeconsuming. Peptide mixtures generated by enzymatic digestion or othermeans from whole cell extracts, organelles, protein complexes, or tissuesamples can contain a extraordinarily large number of peptides. Forexample, a tryptic digest of a whole mammalian cell lysate can contain1,000,000 or more peptides. Analyzing the amount, much less the identityof each peptide in such a mixture is a daunting task. Attempting toidentify the proteins from which the peptides were generated furtherincreases the complexity of the analysis. However, as can be appreciatedby those skilled in the art, a given protein present in a mixture can beidentified and quantified based on one or a few of the peptidesgenerated by digestion of the protein. It is a commonly acceptedpractice in biology to identify the presence of a protein by the bindingof an antibody specific to that protein, even though the antibodyrecognizes and binds to only a small fraction of the total structure ofthe protein. In other words, a protein can be identified by detectingfewer than all of the peptides arising from digestion of the protein.Thus, methods that reduce the complexity of peptide mixtures in acontrolled and predictable manner by isolating a subset of peptidespresent in the mixture can greatly facilitate the identification and/orquantification of the proteins from which the peptide mixture wasgenerated.

[0043] Analysis is facilitated because the time and memory required fordatabase searching to identify the peptides present in the mixture (andthe proteins from which they were derived) is greatly reduced. Theincreases in the speed, simplicity and confidence of analysis that areachieved by the methods of the invention can be realized with, at most,only a minor loss of information. This loss of information can occur,for example, because some small fraction of proteins will, under someconditions, fail to generate peptides that can be tagged with a given Rgroup. Thus, under some circumstances, a small number of proteins willnot be detected. However, the difficulty can be largely overcome byperforming additional analysis. For example, a tagging moiety with adifferent R group can be used in additional analysis. In the case of atagging moiety using a R_(S/T) the proteins in the original mixedprotein sample can be digested in an alternative manner that generatesamino terminal ser peptides and/or amino terminal thr peptides from theproteins that do not generate such peptides under the first set ofdigestion conditions. This highlights one of the strong points of thedescribed method, namely that it relies on sequence information that ispresent in the protein, which means that cutting the protein with anenzyme of different specificity will lead to a set of peptides that canbe nearly orthogonal to the original set. The results from the secondaryanalysis can be combined with the results of the primary analysis tocreate a complete analysis of the proteins present in the originalsample.

[0044] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045]FIG. 1 depicts the distribution of peptides/protein generated bybioinformatic modeling of a trypsin digestion of the entire proteome ofC. elegans.

[0046]FIG. 2 depicts the distribution of cys-containing peptides/proteingenerated by bioinformatic modeling of a trypsin digestion of the entireproteome of C. elegans followed by selection of cys-containing peptides.

[0047]FIG. 3 depicts the distribution of amino terminal-ser and aminoterminal-thr peptides/protein generated by bioinformatic modeling of atrypsin digestion of the entire proteome of C. elegans followed byselection of amino terminal-ser and amino terminal-thr peptides.

[0048]FIG. 4 depicts the structure of biotin hydrazide.

[0049] FIGS. 5A-5C depict various M-L-R tagging moieties.

[0050]FIG. 6 depicts a B-L-Sol-L-R tagging moiety.

DETAILED DESCRIPTION OF THE INVENTION

[0051] Using the methods of the invention complex peptide mixtures canbe simplified by isolating peptides which include particular amino acids(e.g., peptides having an amino terminal serine (“amino terminal-serpeptides”) or an amino terminal threonine (“amino terminal-thrpeptides”)). The peptides are isolated by reacting the peptides with atagging moiety which reacts with the desired peptides and tags them forcapture by a capture reagent. The tagging moiety includes a reactivegroup (R), a linker group (L), and a group (B) that can selectively bindto a capture reagent or, in place of B, a magnetic group (M) thatresponds to a magnetic force. The tagged peptides are captured andisolated either by contacting them with a capture reagent (e.g., acapture reagent bound to a solid support) where the tagging moietyincludes a B group or by attracting the tagged peptide with an appliedmagnetic force where the tagging moiety includes an M group. Dependingon the type of R group used, cysteine-containing peptides,lysine-containing peptides or peptides having either an aminoterminal-serine or an amino terminal-threonine can be isolated fromother materials present in the mixture (e.g., other peptides). Afterisolation, the captured peptides are released from the capture reagentand analyzed by mass spectroscopy. The peptides can be released byselectively cleaving the linker group within the tagging moiety or bydisrupting the interaction between the capture reagent and the groupthat selectively binds to the capture reagent. In many cases thereleased peptides are modified peptides in that they may includecomponents derived from the tagging moiety, e.g., all or part of thelinker group (L) and/or all or part of the reactive group (R). In somecases it may be possible to release the peptide in an essentiallyunmodified form.

[0052] The capture reagent can be avidin or streptavidin or modifiedavidin or strepavidin and the tagging moiety can include biotin or amodified biotin. Alternatively, the capture reagent can be biotin or amodified biotin and the tagging moiety can include avidin orstreptavidin or modified avidin or streptavidin. In order to facilitateisolation of the tagged peptides from other components, the capturereagent can be bound, preferably covalently, to a solid support such asglass particles, the well of a microtiter plate, magnetic particle orthe like. Thus, the tagged peptides can be captured using avidin orstreptavidin coated magnetic particles.

[0053] The peptides, e.g., the amino terminal-ser and amino terminal-thrpeptides, can be generated by cleavage of a protein or a mixture ofproteins. The cleavage can be enzymatic. For example, peptides can begenerated by digestion a protein or mixture of proteins with trypsinusing standard techniques.

[0054] Amino terminal-ser and amino terminal-thr peptides for analysisby mass spectrometry can be prepared as follows. A sample containingmixture of proteins is subjected to denaturing conditions. The denaturedproteins are digested with trypsin. The beta-amino alcohol moietypresent on amino terminal-ser peptides and amino terminal-thr peptidesis selectively oxidized by adding, to the peptide mixture dissolved inpH 7 phosphate buffer, sodium metaperiodate to make the solution 40 mMin periodate. After incubation at room temperature in the dark for 5minutes, the excess oxidant is quenched by the addition of ethyleneglycol. The modified peptides are then biotinylated by adding biotinhydrazide directly to the reaction mixture and incubating for 30minutes. The selectively biotinylated peptides are then captured onmonomeric-streptavidin coated particles. After washing away non-modifiedpeptides and washing with HPLC starting buffer, the peptides are elutedfrom the particles by displacement with free D-biotin. Alternatively, aspecialized biotin-hydrazide that contains a cleavable linker can beused for the biotinylation step and a selective cleavage reagent can beadded to release the bound peptides from the particles. Examples ofcleavable groups that can be incorporated into the linker include adisulfide group (cleaved with TCEP), or a vicinal diol group (cleavedwith sodium periodate). Once the isolated peptides have been releasedfrom the particles, they can be analyzed directly by injecting thesample into the liquid chromatography-mass spectrometry equipment(LC/MS).

[0055] Previously described methods for achieving simplification ofpeptide mixtures have utilized the reactive sulfhydryl group of cysteine(cys) to isolate peptides containing a cys. By isolating peptides havingan amino terminal-ser or an amino terminal-thr, the methods of thepresent invention can result in even greater simplification with littleor no increase in the number of proteins missed. In general, increasedsimplification and knowledge of partial sequence information permits oneto conduct more constrained database searches and results in smallerdatabases with faster searches, requiring less intensive use ofprocessing capacity and memory. Moreover, in the methods of theinvention, a portion of each peptide analyzed is known (e.g., it isknown that there is an amino terminal ser or thr or it is known that acys is present or it is known that a lys is present). This constrainsthe database searching and facilitates the interpretation of MS/MSfragmentation patterns. Moreover, analysis of peptides that are modifiedat the amino terminus may be easier than analysis of internally modifiedpeptides. The methods of the invention reduce the need for peak parkingand hence result in increased sample throughput since occurrence ofco-eluting peaks should decrease.

[0056] The simplification achieved by the present method can beillustrated by examining the proteome resulting from a complex genome.FIGS. 1-3 depict the results of bioinformatic analysis of a trypsindigestion of the entire C. Elegans proteome. FIG. 1 depicts thedistribution of peptides/protein generated by trypsin digestion of theentire proteome. FIG. 2 depicts the distribution of cys-containingpeptides/protein generated by trypsin digestion of the entire proteome.FIG. 3 depicts the distribution of amino terminal-ser and aminoterminal-thr peptides/protein generated by trypsin digestion of theentire proteome. The results of these calculations suggest that thegreater simplification may be achieved by selecting amino terminal-serand amino terminal-thr peptides than by selecting cys-containingpeptides. These calculations suggest that about 5% of the proteome isnot detectable with each approach (the fraction of proteins having 0peptides/protein). These calculations have discarded resulting peptidescontaining 3 or less amino acids as a result of enzymatic cleavage.

[0057]FIG. 4 depicts the structure of biotin hydrazide. This compound isan example of a B-L-R_(S/T)tagging moiety. It includes an B groupcomprising biotin 2 an L group 4, and a R_(S/T) group comprising ahydrazide group 6.

[0058] FIGS. 5A-5C depict exemplary combination of M, L, and R (i.e.,M-L-R tagging moiety). FIG. 5A depicts a tagging moiety suitable forcapture of peptides having an amino terminal Ser or Thr. The taggingmoiety includes a magnetic particle 8, an L group 10 that includes adisulfide bond cleavage site 12, and a reactive group that includes ahydrazide group 14. FIG. 5B depicts a tagging moiety suitable forcapture of peptides having a Cys. The tagging moiety includes a magneticparticle 16, an L group 18 that includes a disulfide bond cleavage site20, and a reactive moiety that includes a malemide group 22. FIG. 5Cdepicts a tagging moiety suitable for capture of peptides having a Lys.The tagging moiety includes a magnetic particle 24, an L group 26 thatincludes a disulfide bond cleavage site 28, and a reactive moiety thatincludes a succinimide group 30.

[0059] The tagging moiety can be isotopically labeled, e.g., bysubstituting one or more atoms in the linker group or the reactivemoiety with a stable isotope of the atom, e.g., one or more hydrogenscan be replaced with deuterium or one or more ¹²C can be replaced with¹³C or ¹⁴N can be labeled with ¹⁵N, or combinations thereof. When anisotopically labeled tagging moiety is used, the released modifiedpeptides will be isotopically labeled. When two peptide samples arereacted with differentially isotopically labeled, but chemicallyidentical, tagging moieties, quantification of the relative amount ofthe peptides in the two samples is facilitated. This is because amixture of the two peptide samples one modified with the “light” form ofthe tag and one modified with the “heavy” form of the tag will contain alight form and a heavy form of two chemically identical entities. Thus,This approach has been used to quantify cys-containing peptides (Gygi etal. (1999) Nature Biotech. 17:994; and PCT Publication WO 00/11208) anda similar approach can be used to quantify amino terminal-ser and aminoterminal-thr peptides and lys-containing peptides.

[0060] Two different peptide samples, e.g., one sample derived fromcells exposed to a selected compound and one sample derived from cellsnot exposed to the selected compound can be differentially isotopicallylabeled using the tagging moieties of the present invention. Theisolated modified differentially isotopically labeled peptides arisingfrom the two samples can be mixed together and analyzed by massspectrometry.

[0061]FIG. 6 depicts an example of a B-L_(A)-Sol-L_(B)-R tagging moiety.The R group of this tagging moiety is biotin 32 the L_(B) group 34 is achain that is at least 13 Å long. A Sol group 36 comprising methoxypolyethylene glycol (MPEG) is connected at one end to the L_(B) group byan amide bond 38 and at the other end to the remainder of the taggingmoiety by an amide bond 40. The MPEG can be as much as or more than5,000 Daltons in mass and can include one or more nucleophilic orelectrophilic groups for reaction with the L group and the R group. TheSol group 36 is connected to the R group 42 by a second linker regionL_(A) 44 that includes a readily cleavable disulfide bond 46. The Rgroup 42 is a succinimide group that can selectively react with aminegroups (e.g., lysine containing peptides).

EXAMPLE

[0062] Bovine serum albumin (BSA; Sigma Chemical, Inc.) and horsemyoglobin (Sigma Chemical, Inc.) were separately digested with trypsin(Promega, Inc.) according to standard procedures. The peptide mixturesobtained from the digestions were treated with NaIO₄. Next excessoxidant was quenched by the addition of ethylene glycol. The modifiedpeptides were then selectively biotinylated by incubating the peptidemixtures with biotin hydrazide (Pierce Chemical, Co.; FIG. 4) for 30minutes. The biotinylated peptides were captured using using MPGstreptavidin-coated magnetic particles (CPG, Inc.). Sample processingwas performed with a KingFisher automated magnetic particle processor(Lab Systems, Inc).

[0063] The samples were analyzed using a Surveyor HPLC (ThermoFinnigan,Inc), configured for nanoflow operation, coupled to a nanospraysource-equipped LCQ Deca mass spectrometer (ThermoFinnigan, Inc.).Reverse phase-HPLC was performed using a PicoFrit packed tip (NewObjective) (75 um i.d. by 10 cm length) and standard reversed-phasegradients at a flow rate of 100 nL/min.

[0064] Based on the sequence of BSA, trypsin digestion should yield sixthr/ser amino terminal peptides. Based on the sequence of horsemyoglobin, trypsin digestion should yield one thr/ser amino terminalpeptide. The six expected peptides for BSA and the one expected peptidefor myoglobin were the only peptides observed in the captured fraction.

[0065] The contents of all references, patents and published patentapplications cited throughout this application are hereby incorporatedby reference.

Other Embodiments

[0066] Two or more tagging moieties with differing R groups can be usedin combination. A tagging moiety that is capable of selectively reactingwith cys-containing peptides can be used to isolate cys-containingpeptides from one fraction of a sample of interest. A tagging moietycapable of selectively reacting with amino terminal-ser and aminoterminal-thr peptides can be used to isolated amino-terminal-ser andamino terminal-thr peptides from a second fraction of the sample ofinterest. The modified peptides isolated using both types of taggingmoieties can be combined and analyzed by mass spectrometry or they canbe independently analyzed and the results combined. If the taggingmoieties can be captured using the same capture reagent, the taggedpeptides (a mixture of cys-containing, amino terminal-ser, and aminoterminal-thr peptides) can be captured with the capture reagent in asingle reaction. The mixture of released, modified peptides can then beanalyzed by mass spectrometry. Differentially isotopically labeledtagging moieties can be used to differentially label the peptides in twoor more different samples. The released peptides can be analyzed bymethods other than mass spectrometry. Thus, the various tagging moietiesand methods of the invention can be used to isolated and purify peptidesor simplify complex mixtures for any purpose.

Equivalents

[0067] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed is:
 1. A method for identifying at least one peptidepresent in a sample, the method comprising: (a) providing a taggingmoiety having the formula: R-L-B wherein R is a reactive group thatreacts with peptides having an amino terminal serine or an aminoterminal threonine, L is a linker group, and B is a group that canselectively binds to a capture reagent; (b) reacting the sample with thetagging moiety to provide tagged peptides; (c) contacting the taggedpeptides with the capture reagent to provide captured tagged peptides;(d) releasing at least the peptide portion of the captured taggedpeptides from the capture reagent to provide released modified peptides;and (e) analyzing the released modified peptides by mass spectrometry.2. The method of claim 1 wherein R comprises □CO—NH—NH₂.
 3. The methodof claim 1 wherein B comprises biotin.
 4. The method of claim 1 whereinB comprises d-iminobiotin.
 5. The method of claim 3 wherein the capturereagent comprises avidin or streptavidin.
 6. The method of claim 3 wherein the releasing step comprises exposing the captured taggedpolypeptides to biotin.
 7. The method of claim 3 wherein the capturereagent comprises an antibody selective for biotin.
 8. The method ofclaim 1 wherein the tagging moiety is biotin hydrazide.
 9. The method ofclaim 1 wherein the sample is exposed to an oxidizing agent prior toexposing the sample to the tagging moiety.
 10. The method of claim 1wherein L comprises a disulfide group.
 11. The method of claim 1 whereinL comprises a vicinal diol group.
 12. The method of claim 1 wherein L isisotopically labeled.
 13. The method of claim 1 wherein the releasedmodified peptides are separated by chromatography prior to analysis bymass spectrometry.
 14. The method of claim 1 wherein R is isotopicallylabeled.
 15. The method of claim 1 wherein the analysis by massspectrometry comprises identifying at least one peptide.
 16. The methodof claim 1 wherein the analysis by mass spectrometry comprisesquantifying at least one peptide.
 17. A reagent kit comprising: atagging moiety having the formula: R-L-B wherein R is a reactive groupthat reacts with peptides having an amino terminal serine or an aminoterminal threonine, L is a linker group, and B is a group that canselectively bind a capture reagent.
 18. The reagent kit of claim 17further comprising a proteolytic enzyme.
 19. The reagent kit of claim 17further comprising a capture reagent.
 20. The reagent kit of claim 17wherein R comprises —CO—NH—NH₂.
 21. The reagent kit of claim 17 whereinthe tagging moiety is biotin hydrazide.
 22. The reagent kit of claim 21further comprising a capture reagent comprising avidin or streptavidinbound to a solid support; and D-biotin.
 23. The reagent kit of claim 22further comprising an oxidizing agent.
 24. The reagent kit of claim 23wherein the oxidizing agent is sodium metaperiodate and the kit furthercomprises an agent capable of quenching the oxidizing agent.
 25. Thereagent kit of claim 17 wherein L or R is isotopically labeled.
 26. Amethod for identifying at least one peptide present in a sample, themethod comprising: (a) providing a tagging moiety having the formula:R-L-M wherein R is a reactive group that reacts with peptides comprisinga selected amino acid, L is a linker group, and M is a magnetic particlethat can be attracted by a magnetic force; (b) reacting the sample withthe tagging moiety to provide tagged peptides; (c) isolating the taggedpeptides by applying a magnetic force that attracts M; (d) releasing atleast the peptide portion of the captured tagged peptides from the Mgroup to provide released modified peptides; and (e) analyzing thereleased modified peptides by mass spectrometry.
 27. The method of claim26 wherein L is isotopically labeled.
 28. A reagent kit comprising: atagging moiety having the formula: R-L-M wherein R is a reactive groupthat reacts with peptides comprising a selected amino acid, L is alinker group, and M is a magnetic particle that can be attracted by amagnetic force.
 29. The reagent kit of claim 28 wherein R comprises: (a)an amine reactive moiety, (b) —CO—NH—NH₂. (c) a thiol reactive moiety,or
 30. The reagent kit of claim 28 wherein L is isotopically labeled.31. The reagent kit of claim 28 wherein R is isotopically labeled.
 32. Amethod for identifying at least one peptide present in a sample, themethod comprising: (a) providing a tagging moiety having the components:R, L, Sol and M wherein R is a reactive group that reacts with apeptides comprising a selected amino acid, L is a linker group, Sol is ahydrophilic polymer, and M is a magnetic particle that can be attractedby a magnetic force; (b) reacting the sample with the tagging moiety toprovide tagged peptides; (c) contacting the tagged peptides with thecapture surface that can magnetically bind to the magnetic particle toprovide captured tagged peptides; (d) releasing at least the peptideportion of the captured tagged peptides from the capture surface toprovide released modified peptides; and (e) analyzing the releasedmodified peptides by mass spectrometry.
 33. A reagent kit containing amoiety comprising: a tagging moiety having the formula R-L-Sol-MR-L-Sol-L-M or R-Sol-L-M, wherein R is a reactive group that reacts witha peptides comprising a selected amino acid, L is a linker group, Sol isa hydrophilic polymer, and M is a magnetic particle that can beattracted by a magnetic force.
 34. The reagent kit of claim 33 where Rcomprises: (a) an amine reactive moiety, (b) a thiol reactive moiety,(c) —CO—NH—NH₂; or (d) an enzymatic substrate or other noncovalentelement of molecular recognition such as ATP, GTP, NAD, NADP, NADH,NADPH, ubiquitin, or structural analogs thereof.
 35. The kit of claim 33wherein R or L is isotopically labeled
 36. A method for identifying atleast one peptide present in a sample, the method comprising: (a)providing a tagging moiety having the components: R, L, Sol and Bwherein R is a reactive group that reacts with a peptides comprising aselected amino acid, L is a linker group, Sol is a hydrophilic polymer,and B is a group that can selectively bind a capture reagent; (b)reacting the sample with the tagging moiety to provide tagged peptides;(c) contacting the tagged peptides with the capture surface that canmagnetically bind to the magnetic particle to provide captured taggedpeptides; (d) releasing at least the peptide portion of the capturedtagged peptides from the capture surface to provide released modifiedpeptides; and (e) analyzing the released modified peptides by massspectrometry to identify at least one peptide present in the sample. 37.A reagent kit containing a moiety comprising: a tagging moiety havingthe formula R-L-Sol-B or R-Sol-L-B, wherein R is a reactive group thatreacts with a peptides comprising a selected amino acid, L is a linkergroup, Sol is a hydrophilic polymer, and B is a group that canselectively bind to a capture reagent.
 38. The reagent of claim 37 whereR comprises: (a) an amine reactive moiety, (b) a thiol reactive moiety,(c) —CO—NH—NH₂; or (d) an enzymatic substrate or other noncovalentelement of molecular recognition such as ATP, GTP, NAD, NADP, NADH,NADPH, ubiquitin, or structural analogs thereof.
 39. The reagent kit ofclaim 37 wherein R or L is isotopically labeled
 40. A reagent kitcontaining a moiety comprising: a tagging moiety having the formulaR-L-Sol, wherein R is a reactive group that reacts with a peptidescomprising a selected amino acid, L is a linker group, and Sol is ahydrophilic polymer.
 41. The reagent kit of claim 40 where R comprises:(a) an amine reactive moiety, (b) a thiol reactive moiety, (c)—CO—NH—NH₂; or (d) an enzymatic substrate or other noncovalent elementof molecular recognition such as ATP, GTP, NAD, NADP, NADH, NADPH,ubiquitin, or structural analogs thereof.
 42. The kit of claim 37wherein R or L is isotopically labeled.
 43. A method for analyzing aportion of a polypeptide present in a sample, the method comprising: (a)providing a tagging moiety having the formula: R-L-M wherein R is areactive group that reacts with polypeptides comprising a selected aminoacid, L is a linker group, and M is a magnetic particle that can beattracted by a magnetic force; (b) reacting the sample with the taggingmoiety to provide a tagged polypeptide; (c) isolating the taggedpolypeptide by applying a magnetic force that attracts M to provide anisolated tagged polypeptide; (d) enzymatically digesting the isolatedtagged polypeptide to provide an isolated tagged polypeptide fragment;(e) releasing at least the polypeptide fragment portion of the isolatedtagged polypeptide fragment from the M group to provide a releasedmodified polypeptide fragment; and (f) analyzing the released modifiedpolypeptide fragment by mass spectrometry.
 44. A method for analyzing aportion of a polypeptide present in a sample, the method comprising: (a)providing a tagging moiety having the formula: R-L-M wherein R is areactive group that reacts with polypeptides comprising a selected aminoacid, L is a linker group, and M is a magnetic particle that can beattracted by a magnetic force; (b) reacting the sample with the taggingmoiety to provide a tagged polypeptide; (c) digesting the taggedpolypeptide provide an tagged polypeptide fragment; (d) isolating thetagged polypeptide fragment by applying a magnetic force that attracts Mto provide an isolated tagged polypeptide fragment; (e) releasing atleast the polypeptide fragment portion of the isolated taggedpolypeptide fragment from the M group to provide a released modifiedpolypeptide fragment; and (f) analyzing the released modifiedpolypeptide fragment by mass spectrometry.
 45. A method for analyzing aportion of a polypeptide present in a sample, the method comprising: (a)providing a tagging moiety having the formula: R-L-B wherein R is areactive group that reacts with polypeptides comprising a selected aminoacid, L is a linker group, and B is a group that can selectively bind toa capture reagent; (b) reacting the sample with the tagging moiety toprovide a tagged polypeptide; (c) contacting the tagged polypeptidefragment with the capture reagent to provide a captured taggedpolypeptide; (d) digesting the captured tagged polypeptide to provide acaptured tagged polypeptide fragment; (e) releasing at least thepolypeptide portion of the capture tagged polypeptide fragment from theB group to provide released modified polypeptide fragment; and (f)analyzing the released modified polypeptide fragment by massspectrometry.
 46. A method for analyzing a portion of a polypeptidepresent in a sample, the method comprising: (a) providing a taggingmoiety having the formula: R-L-B wherein R is a reactive group thatreacts with polypeptides comprising a selected amino acid, L is a linkergroup, and B is a group that can selectively binds to a capture reagent;(b) reacting the sample with the tagging moiety to provide a taggedpolypeptide; (c) digesting the tagged polypeptide provide an taggedpolypeptide fragment; (d) contacting the tagged polypeptide fragmentwith the capture reagent to provide a captured tagged polypeptidefragment; (e) releasing at least the polypeptide fragment portion of thecaptured tagged polypeptide fragment from the B group to provide areleased modified polypeptide fragment; and (f) analyzing the releasedmodified polypeptide fragment by mass spectrometry.
 47. The method ofany of claims 43-46 wherein L is isotopically labeled.
 48. The method ofany of claims 43-46 wherein R is isotopically labled.